A partially purified human factor IX was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 9 were injected separately into two rabbits, R1 and R2. On immunodiffusion R1 antiserum showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and slightly factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, this antiserum revealed two new precipitating contaminants. The R2 antiserum neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera R1 and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX. We now plan: 1) to develop quantitative assays for the factor IX protein using R2 antiserum, either by Laurell's technique or, if insensitive, by radioimmunoassay; 2) to quantitate factor IX antigen in the 90 Hemophilia-B plasmas we have collected, in parallel with neutralization studies; 3) to study in the same way obligatory and suspected carriers of the Hemophilia-B gene, patients with advanced liver disease, on oral anticoagulants and newborns; 4) to purify factor IX by immunoabsorption; 5) to develop crossed immunoelectrophoresis using antiserum Rs; 6) to study antigenic changes observed during trypsin activation of factor IX.